Characterization of ColA as a virulence factor of Vibrio alginolyticus H1 pathogenic to cuttlefish Sepia pharaonis

Abstract

Vibrio alginolyticus is a marine opportunistic bacterium that can infect a variety of aquatic animals. In this study, V. alginolyticus H1 was found to degrade collagen from the skin of the cuttlefish Sepia pharaonis, therefore; a colA gene encoding collagenase was cloned from V. alginolyticus H1. The expression at the mRNA level and enzymatic activity of ColA under different redox pressures were determined using real time RT–PCR and gelatin biochemical tubes. The catalytic domain of ColA was recombined in Escherichia coli BL21(DE3), recombinant ColA-CD (rColA-CD) was purified via Ni-NTA agarose, and the optimum temperature and pH for rColA-CD were approximately 40 °C and 9.0, respectively. To further investigate the role of ColA in bacterial virulence, the colA gene was knocked out to construct the ΔcolA mutant, which exhibited reduced virulence to S. pharaonis compared with that of the wild type (WT) strain. Transcriptomic analysis revealed that, compared with the cuttlefish S. pharaonis infected with WT, the individuals infected with ΔcolA showed 392 differentially expressed genes (DEGs), including 111 upregulated DEGs and 281 downregulated DEGs, among which the level of mammalian matrix metalloproteinase 19 was downregulated. Immune-related pathways, such as the ErbB signaling pathway, and JAK-STAT signaling pathway were downregulated, whereas protein processing in the endoplasmic reticulum was upregulated. This study demonstrated that the collagenase encoded by the colA gene is a critical virulence factor of V. alginolyticus, which regulates specific immune-related pathways in the cuttlefish S. pharaonis.